Identification and expression analysis of YABBY family genes in Platycodon grandiflorus

ABSTRACT Platycodon grandiflorus set ornamental, edible, and medicinal plant with broad prospects for further application development. However, there are no reports on the YABBY transcription factor in P. grandiflorus. Identification and analysis of the YABBY gene family of P. grandiflorus using bioinformatics means. Six YABBY genes were identified and divided into five subgroups. Transcriptome data and qRT-PCR were used to analyze the expression patterns of YABBY. YABBY genes exhibited organ-specific patterns in expression in P grandiflorus. Upon salt stress and drought induction, P. grandiflorus presented different morphological and physiological changes with some dynamic changes. Under salt treatment, the YABBY gene family was down-regulated; PgYABBY5 was up-regulated in leaves at 24 h. In drought treatment, PgYABBY1, PgYABBY2, and PgYABBY3 were down-regulated to varying degrees, but PgYABBY3 was significantly up-regulated in the roots. PgYABBY5 was up-regulated gradually after being down-regulated. PgYABBY5 was significantly up-regulated in stem and leaf at 48 h. PgYABBY6 was down-regulated at first and then significantly up-regulated. The dynamic changes of salt stress and drought stress can be regarded as the responses of plants to resist damage. During the whole process of salt and drought stress treatment, the protein content of each tissue part of P grandiflorus changed continuously. At the same time, we found that the promoter region of the PgYABBY gene contains stress-resistant elements, and the regulatory role of YABBY transcription factor in the anti-stress mechanism of P grandiflorus remains to be studied. PgYABBY1, PgYABBY2, and PgYABBY5 may be involved in the regulation of saponins in P. grandiflorus. PgYABBY5 may be involved in the drought resistance mechanism in P. grandiflorus stems and leaves. This study may provide a theoretical basis for studying the regulation of terpenoids by the YABBY transcription factor and its resistance to abiotic stress.


Introduction
Platycodon grandiflorus is a perennial herb of the Platycodon genus in the Platycodon family. It is mainly distributed in Northeast China, North Korea, Japan, Russia, etc., and is used as an auxiliary ingredient in dishes or used directly in pickles. At present, more than 100 kinds of compounds, such as steroidal saponins, flavonoids, phenolic acids, polyacetylenes, and sterols, have been isolated from P. grandiflorus. Triterpenoid saponins are the main active components in P. grandiflorus, including platycodins D, E, A, etc. It is one of the main triterpenoids 1 . which have many pharmacological benefits, such as enhancing immune stimulation, antiinflammation, anti-obesity, anti-atherosclerosis, and anticancer effects. In addition, P. grandiflorus has been used to treat many chronic diseases, such as bronchitis, asthma, and tuberculosis. 2 P. grandiflorus polysaccharides (PGPs) are another important active ingredient in this species, which are related to the antioxidant activity; for instance, selenium polysaccharides in P. grandiflorus may be a potentially useful antioxidant. 3,4 In addition, PGPs can activate macrophages and enhance nonspecific immune functions. 5 YABBY is a family of unique transcription factors in plants, which has two highly conserved domains in its members: the zinc finger-like domain and the YABBY domain. [6][7][8] The zinc finger domain is located at the N-terminal of the YABBY protein, and the YABBY domain is located at the C-terminal. The zinc finger structure belongs to the C2C2 type, and there are gaps of 20 amino acids between cysteine pairs. The YABBY domain has a helix-loop-helix structure, 4,9 which constitutes the first two of the three α helices in the high mobility group (HMG) box. Based on the different expression patterns of this gene in angiosperms, it can be divided into two types: the vegetative and reproductive types. 10,11 The 'vegetative' genes regulate the polar development of lateral organs, formation of edges, the growth of meristem and leaves at the stem tips, and the maturation of leaves. [12][13][14] The reproductive type includes CRABS CLAW (CRC) and INNER-NO-OUTER (INO), which are only expressed in developing carpels and ovules, respectively. 15,16 According to the phylogenetic analysis of YABBY proteins in different species, the six YABBY members in Arabidopsis thaliana: FILAMENTOUS FLOWER (FIL), YABBY3 (YAB3), CRABS CLAW (CRC), INNER NO OUTER (INO), YABBY2 (YAB2), andYABBY5. [17][18][19] In a study on A.thaliana, FIL, YABBY2, YABBY3, and YABBY5 were observed to be expressed in both vegetative and reproductive organs. 20 The YABBY gene has been established to play an important role in the development and growth of plants. An FIL, OsYABBY4, is mainly expressed in rice vascular tissues to regulate vascular development, 21 while YABBY2 member in Oryza sativa, OsYABBY1, FIL/YAB3 members in Zea mays, ZYABBY9 and ZYABBY14, and YABBY2, YABBY3, and YABBY5 in A. thaliana have redundant functions to promote the development of collateral organs. 13,22 CRC is restricted to the developing carpel and nectaries in A. thaliana. 15 ZmYABBY1 and ZmYABBY11 regulate the development of male florets, 23 while drooping leaves in rice regulate the development of carpel and the formation of the midvein. 24 CRC members in rice and corn also have conservative functions in leaf development, whereby they affect leaf width, length, angle, and internode diameters. 25,26 INO promotes the development of ovule ectoderm into the seed coat. 15 In A. thaliana, INO participates in the formation and development of the exocarp. 27 YABBY genes are also involved in plant hormone responses. For example, overexpression of OsYABBY1 in rice leads to a semidwarf phenotype through the feedback regulation of gibberellin (GA), as well as its biosynthesis and metabolism; 28 OsYABBY4 regulates plant development and growth by regulating GA signaling pathways. 21 YABBY genes are also involved in abiotic stress. For example, over-expression of AcYABBY4 from pineapple in A. thaliana can negatively regulate the salt tolerance of plants. 29 Genome-wide analysis of the YABBY gene in kidney beans showed that they were involved in salt stress; 30 similarly, GmYABBY10 in soybean was noted to negatively regulate drought and salt tolerance in plants. 31 For example, molecular cloning results presented by Takahiro Yamaguchi and other researchers have shown that DL is a member of the YABBY gene family that is closely related to CRC in A. thaliana based on evolutionary relationships. During the process of rice flower development, carpel development is regulated by DL. When the function of DL is seriously deficient, a complete homeotic transformation 28 from carpel to stamen occurs, without affecting the characteristics of other flowering organs.
This study presents findings from bioinformatics analysis of the YABBY gene family in P. grandiflorus. The dynamic changes in the differences in PgYABBY expression were analyzed under salt and drought stress, laying a foundation for the study of YABBY transcription factors that may be involved in the regulation of terpenoids and in imparting resistance against abiotic stress in P. grandiflorus. Our results will provide theoretical support for follow-up studies on secondary metabolites of medicinal plants to optimize the breeding of P. grandiflorus varieties.

Materials
The whole genome data of P grandiflorus (GCA_016624345.1) comes from NCBI (https://www.ncbi.nlm.nih.gov/) database. It is acquired from the 4th generation inbred of Jangbaek-doraji. The YABBY protein sequence information for A. thaliana (GCA_000005425.2) was obtained from the Plant TFDB (http://planttfdb.cbi.pku.edu.cn/) database, while the transcriptome data of the different tissue parts were obtained by self-test.

Identification and analysis of PgYABBY
Repeated transcripts caused by variable shearing were removed to obtain the member ID of the YABBY gene family. The protein sequences of the YABBY family proteins from P. grandiflorus were then obtained by TB tools 32 software. The size, relative molecular weight, theoretical isoelectric point, and hydrophilic average coefficient of the YABBY protein sequence of P. grandiflorus were predicted on the ExPASy website (https://web.expasy.org/protparam/). WoLFPSORT website (http://www.genscript.com/wolf-psort.html) was used to predict the subcellular localization of YABBY family members. (Supplementary Table 1). Homology modeling techniques are widely used in protein models. To determine the tertiary structure of YABBY protein, we used the fully automated protein structurehomology modeling server Phyre2 database (http:// www.sbg.bio.ic.ac.uk/phyre2) for 33 homology modeling.

Multiple sequence comparison and phylogenetic analysis of PgYABBY Protein
Multiple sequence alignment of all identified YABBYs in P. grandiflorus was carried out using ClustalW, 34 and a phylogenetic tree was generated by neighbor-joining (NJ) method with default parameters: bootstrap method setting to 1000, Poisson model, and complete deletion in MEGA 11.The conserved domain of a YABBY protein, PgYABBY, in P. grandiflorus was compared and analyzed by using DNAMAN software. The data of this YABBY protein for A. thaliana were obtained from the Plant TFDB database, which was then merged with the file of the YABBY protein of P. grandiflorus. Multi-sequence alignment was carried out by the MUSCLE program. The neighbor-joining (NJ) method was performed using MEGA11 35 software with a Bootstrap value of 1000 repetitions to construct a phylogenetic tree.

Analysis of PgYABBY gene structure and conserved motif
TB tools were used to analyze YABBY gene structure, and online software, MEME (http://meme-suite.org/tools/meme), was used to predict the motif, where the maximum motif number was set to 10. After setting other default parameters, TB tools was used to visualize the genetic structure and motifs in the evolutionary tree.

Prediction of PgYABBY Cis-acting elements
TB tools was used to extract the 2000-bp sequence upstream of the target YABBY gene in P. grandiflorus, considering it the promoter region. (Supplementary Table 2) The sequence was submitted to Plant Care (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) as predicted cis-acting elements. the result files were screened, classified, and visualized.

Analysis of PgYABBY gene expression
The transcriptome data of leaves, petals, pistils, roots, seeds, sepals, stamens, and stems of Landrace Doraji were standardized, followed by cluster analysis. The heat map was drawn using TB tools.

cDNA preparation
Total Plant RNA Kit (product serial number 5101050) was purchased from Hangzhou Xinjing Biochemical Reagent Development Co., Ltd. (Hangzhou China) The first strand cDNA High-Efficiency Synthesis (Reverse Transcription) Kit (article number QP057) was purchased from Guangzhou Yijin Biotechnology Co., Ltd. (Guangzhou China). RNA was extracted according to the methods and steps described by Hangzhou Xinjing Biochemical Reagent Development Co., Ltd. After extracting RNA, it was stored at −80°C. For analysis, a proper amount of RNA was the reverse transcription reagent was melted while being kept on ice. The reverse transcription program was applied as follows: 25°C for 5 min, 42°C for 15 min, 85°C for 5 min, and 4°C for the holding step. The concentration of cDNA products was measured by an ultramicro ultraviolet spectrophotometer, and cDNA was stored in the refrigerator at −20°C for later use.

Real-time quantitative experiment
BlazeTaqTMSYBR®GreenqPCRMix2.0 Real-Time Fluorescence Quantitative PCR Detection Reagent (article number QP031) was purchased from Guangzhou Yijin Biotechnology Co., Ltd. According to the laboratory instructions of the reagent company. In order to study the expression patterns of PgYABBY genes under stress, a real-time quantitative fluorescence test was performed on the annually bred P. grandiflorus that had been subjected to different treatments. cDNA was diluted according to the experimental requirements, and real-time quantitative PCR was performed using the manufacturer's instructions. PCR on each sample was repeated three times. PrimerPrimer5 software was used to design primers, and the Tm value was kept between 55°C and 60°C. PCR was performed to verify whether the primers were effective. Primers used in the P. grandiflorus expression pattern experiment were synthesized by Harbin Qingke Jiamei Biotechnology Co., Ltd. Table 3).

Basic information on YABBY Genes in P. grandiflorus
Each YABBY gene in P. grandiflorus was given a transcript accession number. Using basic information on the YABBY gene family in P. grandiflorus, it was found that their sizes are between 183 and 277 aa. The proteins encoded by PgYABBY genes are predicted to be located in the nucleus, their isoelectric points were between 4.72 and 9.38, while their relative molecular weights were different. YABBY proteins are hydrophilic with different hydrophilicities, and the aliphatic amino acid index was observed to be between 61.86 and 77.42. (Table 1) The tertiary structure analysis of P. grandiflorus YABBY gene shows that the members of the subfamily are similar in structure, and the proteins containing the helix domain bound to CCDC124-80S-eERF1 ribosome complex are highly similar. There are also differences in the structure of different subfamily genes, For example, different proteins have different structures ( Figure 1) The diagram shows the tertiary structure of six YABBY proteins from three subfamilies.

Phylogenetic analysis and classification
To better explore the evolutionary relationship of YABBYs, six Arabidopsis YABBYs,eight rice YABBYs, nine Solanum lycopersicum YABBYs,thirteen Zea mays YABBYs, eleven Brassica oleracea YABBYs, twelve Pak-choi YABBYs, seven Ananas comosus YABBYs, and six YABBY members in P. grandiflorus were used to construct an evolutionary tree using MEGA 11 with ClustalW and NJ methods. As reported in Arabidopsis, the P. grandiflorus YABBYs were also divided into five subfamilies, YAB1, YAB2, CRC, INO, and YAB5 ( Figure 2). The FIL and YAB2 subfamilieshad the larger numbers of YABBYs, in which the FIL subfamily contained 24 members while YAB2 subfamily contained 17 members. Subfamilies of CRC shared the smallest YABBYs with nine members, respectively. P. grandiflorus YAB1, YAB2 and YAB5 are all divided into YAB5 subfamily, YAB4 and YAB6 are all distributed in INO subfamily, and finally only YAB3 belongs to FIL subfamily ( Figure 1 and Table 1). Taken together, these results suggested that there are evolutionary splits and diversifications of YABBYs among different species.
In MEGA 11, phylogenetic trees without roots were developed using the neighbor-joining (NJ) process. At each node, bootstrap values of 1,000 replicates are indicated. Dark gray triangle represents the YABBY sequence of A.thaliana, green right pointing triangle represents the YABBY sequence of Oryza sativa, red circle represents the YABBY sequence of Solanum lycopersicum, yellow left pointing triangle represents the YABBY sequence of Zea mays, black star represents the YABBY sequence of Brassica oleracea, green star represents the YABBY sequence of Pak-choi, blue square represents the YABBY sequence of Ananas comosus, and finally purple square represents the YABBY sequence of P. grandiflorus. (Figure 2)

Relationship between PgYABBY Protein sequence alignment and evolution
PgYABBY1, PgYABBY3, PgYABBY4, PgYABBY5, and PgYABBY6 all have complete conservative domains, and PgYABBY2 contains only some conservative amino acids, such as valine, cysteine, and glycine in the zinc finger domain, arginine, glutamic acid, proline, and aspartic acid in the YABBY domain ( Figure 3).

Conserved motifs and gene structure in the PgYABBY protein
A total of 10 conserved motifs were found in the YABBY proteins of P. grandiflorus (Supplementary Figure 1), among which Motif1 ( Figure 4a) and Motif2 ( Figure 4b) were highly conserved. Each protein contained Motif2, and all proteins, except PgYABBY2, contained Motif1. The conserved amino acids of Motif1 include proline, glutamic acid, lysine, arginine, serine, alanine, and phenylalanine, while those of Motif2 include valine, serine, proline, threonine, cysteine, glycine, histidine, and leucine. According to the evolutionary relationships among PgYABBY proteins ( Figure 5), it can be inferred that closely related proteins have similarly conserved motifs (Figure 5a), For instance, PgYABBY4 and PgYABBY6, which belong to the INO subfamily contain Motif1, Motif2, Motif3, and Motif9, where the motifs are arranged in the same order. PgYABBY1 and PgYABBY2 both contain Motif2, Motif5, and Motif7, while PgYABBY1 and PgYABBY5 both contain Motif1 and Motif2. PgYABBY2 and PgYABBY5 both contain Motif8. PgYABBY3 contains three conserved motifs, including Motif6, in addition to the two highly conserved motifs.
Visualizations of the structure of the YABBY genes in P. grandiflorus using TB Tools software showed that PgYABBY2 has two very special introns, while other YABBY   gene structures were conserved. The number of introns was generally 6-7, of which only PgYABBY4 contained seven introns. The introns were extremely long, and the rest of the members contained only six introns. (Figure 5b)

Prediction of Cis-elements of the PgYABBY gene
The promoter region of the YABBY genes in P. grandiflorus contained a variety of cis-elements ( Figure 6). Each YABBY gene contained light-responsive elements, which constitute the largest number of elements, followed by some hormonal cis-elements. PgYABBY1 contained the greatest number of different kinds of cis-elements, including 11 kinds. These specifically include elements dealing with defense and stress response, zein metabolism regulation, and endosperm expression. PgYABBY2 contained five cis-acting elements, most of which are photo-response elements. The PgYABBY3 promoter region contained six elements. PgYABBY4 had four cis-acting elements, where the number of abscisic acid-responsive elements was second only to that of photo-response elements. PgYABBY5 and PgYABBY6 contained only three cis-acting elements, both of which contained lightresponsive elements and abscisic acid-responsive elements.

Expression Pattern of the PgYABBY gene
From the heat map (Figure 7), we can infer that the expression of PgYABBY1 in sepals, leaves, pistils, and petals was higher than that in other tissue parts. The FPKM values in petals, pistils, and sepals were all higher than 6.0. PgYABBY1, PgYABBY2, and PgYABBY5 are homologous genes. PgYABBY2 was only slightly expressed in various tissue parts, and the highest expression level was recorded in the roots. PgYABBY5 was very similar to PgYABBY1 in its expression pattern, but it was only weakly expressed in roots and seeds. The expression of PgYABBY3 in pistil was the highest with an FPKM value of 8.50, followed by sepals, where the FPKM value was 7.31. PgYABBY4 was not expressed in all tissues. The expression of PgYABBY6 in sepals was ~8 times higher than that in seeds, but there was no expression in other tissues.  The changing trend of PgYABBY2 in the three tissue parts under salt stress was basically the same, and the expression level was down-regulated as a whole. At the root, PgYABBY2 was down-regulated within 24 h and was up-regulated at 48 h after treatment. In the stem, PgYABBY2 decreased significantly after 12 h of treatment and then increased slightly. In the middle leaves, PgYABBY2 decreased significantly at 12 and 24 h and increased at 48 h. Salt stress also inhibited the expression of PgYABBY3 in P. grandiflorus. In the root, PgYABBY3 was down-regulated and slightly up-regulated. In the stem, PgYABBY3 showed an upward trend at 24 h after  being strongly down-regulated, and then was down-regulated finally. PgYABBY3 was down-regulated in the leaves at first and then increased after 48 h.
At the root, PgYABBY4 increased at first followed by a significant decrease at 24 h, and then increased significantly at 48 h. Salt stress had little effect on the expression of PgYABBY4 in stems, and its expression reached the lowest level after 24 h of salt stress. In the leaves, PgYABBY4 decreased to the lowest level at 12 h and then increased continuously. Under the influence of salt stress, root PgYABBY5 was downregulated, although it had an upward trend in 24 h; however, the amplitude remained small. PgYABBY5 was down-regulated in the middle stem. PgYABBY5 showed a significant downward trend at 12 h in leaves, then increased significantly, and finally decreased sharply at 48 h. Salt stress has a strong influence on the expression of PgYABBY6. After 12 h of treatment, the expression of PgYABBY6 in root was significantly upregulated, then down-regulated at 24 h, and then finally upregulated. In the stem, the expression of PgYABBY6 was downregulated; it slightly increased at 24 h and then decreased to the lowest level at 48 h. In the middle leaves, PgYABBY6 decreased significantly at 12 h, then increased significantly, and remained stable till the 48th hour. After salt stress treatment, the expression pattern of the YABBY genes in different tissue parts was analyzed. It was found that the expression of the YABBY genes in the three different tissues, root, stem, and leaf, changed with a change in stress treatment time, indicating that the YABBY gene can play different roles and functions in coping with abiotic stress in P. grandiflorus.

Analysis of expression pattern of YABBY gene under drought stress
Drought stress can obviously inhibit the expression of PgYABBY1. The expression of PgYABBY1 in roots, stems and leaves was down-regulated, especially in stems, which was down-regulated by about 650 times at 48 h.
The expression of PgYABBY2 was also inhibited by drought stress; it always showed a downward trend in roots, stems, and leaves, especially when the leaves were treated for 24 h. In the roots, with an increase in exposure time to drought stress, PgYABBY3 generally showed an upward trend, and it significantly increased at 48 h. Drought stress inhibited the expression of PgYABBY3 in stems, where its expression levels decreased continuously. In leaves, PgYABBY3 was downregulated first and then up-regulated. During the whole treatment process, the expression of PgYABBY4 was not obvious; its expression level in roots and stems was continuously downregulated. In leaves, PgYABBY4 was down-regulated at 12 h and then up-regulated. Drought stress inhibited the expression of PgYABBY5 in roots; its expression showed a downward trend during treatment. PgYABBY5 was slightly expressed in the stem at first and was up-regulated after 48 h of exposure to drought. The expression level of PgYABBY5 in middle leaves was low from 12 to 24 h, and it was up-regulated by about 3 times at 48 h. The PgYABBY6 gene was significantly upregulated in roots with change in treatment time; it was upregulated by nearly 250 times at 12 h, down-regulated at 24 h, and then increased again. PgYABBY6 was significantly downregulated at 12 h in the stem, then up-regulated continuously. It was up-regulated ~20 times at 48 h. In leaves, PgYABBY6 was first up-regulated and then down-regulated, and then upregulated by ~4 times. Therefore, different observations were made on the YABBY genes in different tissue parts under drought stress. It was found that the expression of the YABBY genes in the three different tissue parts, root, stem, and leaf, can change with the change in exposure time (Figure 9), indicating that the YABBY genes can play different roles in coping with drought stress in P. grandiflorus.

Discussion
In this study, a total of six YABBY genes were identified in P. grandiflorus. Each P. grandiflorus gene was assigned a transcript accession number, and the number of amino acids encoded by them was between 183 and 277, which is similar to the number of amino acids encoded by the YABBY genes reported in other species, such as Arabidopsis. 29 Except for PgYABBY2, which had only a few conserved amino acids, all P. grandiflorus YABBY proteins featured YABBY and zinc finger domains. 36 Two highly conserved motifs were found in the YABBY proteins, which were speculated to be related to the conserved domains of the YABBY transcription factor and the YABBY and zinc finger domains. PgYABBY2 contained only one conserved motif, rendering it different from other PgYABBY proteins, but the reason for this difference remains unclear. The number of introns in the PgYABBY genes was relatively conserved, and only the PgYABBY2 gene presented a special structure. Other PgYABBY genes were consistent with the reported YABBY genes in different species, 36 indicating that the YABBY genes are conserved in different species.
The upstream promoter region of P. grandiflorus contained a variety of cis-acting elements, among which light-and abscisic acid-responsive elements were distributed in each P. grandiflorus YABBY gene. The light-responsive elements were more numerous, indicating that the YABBY genes may be affected by light during the growth and development of P. grandiflorus. PgYABBY1 contained the most abundant cisacting elements, such as some hormones and developmentrelated elements. In addition, it specifically contains three elements: those related to defense, stress response, zein metabolism regulation, and endosperm expression, which indicates that PgYABBY1 may be related to stress resistance and developmental regulation in plants. The elements related to meristem expression exist only in PgYABBY1 and PgYABBY2, indicating that these two genes may be involved in inducing meristem expression. which suggesting that these genes might be involved in different developmental processes, which was consistent with the study by Dai et al. 37 Genes containing drought-inducing elements may be involved in plants'resistance to drought stress.
YABBY genes can act as pivotal regulatory factors involved in the leaf evolution in plants. They regulate various developmental processes, such as restriction of meristem apex, polarity, layered development, the establishment of leaf margin, flower differentiation, carpel formation, outer carpel growth, inflorescence, etc. 8,38 The YABBY transcription factor gene plays an indispensable regulatory role in the formation of lateral organs of plant, while boosting their development. 39 It was found that the YABBY family members play a key role in the development of leaves and leaf-derived organs, such as cotyledons and flowers. 40 Gene structure is always conserved during the process of evolution. 41,42 Our results showed that the density of splicing of the regulatory sequences increased with the increase in intron length (<1.5 kb), and the increase in intron length (>1.5 kb) was related to the increase in splicing site strength. 43 The intron patterns across the different genes may play an evolutionary role in expression conservation or splicing modulation in P. grandiflorus. Furthermore, the intron burden of evolutionarily conserved genes has been reported to be large, and the degree of evolutionarily conserved eukaryotic genes is positively correlated with the size of the intron region. 44 Similarly, the range of expression of FIL/YABBY3-like, YABBY2-like, and YABBY5-like genes in Myrica rubra was wider than that of CRC-like and INO-like genes, which may take part in more significant biological functions, which is consistent with previous studies on A. thaliana. 13 Cis-elements in the promoter region play an important role in gene expression regulation. The presence or absence of these elements affects gene expression. 45 The conserved cis-acting motif can be used to forecast the function and underlying interaction of genes. 46 The conserved cis-acting motif can be used to forecast the function and underlying interaction of genes. 10,13 In order to better comprehend the regulation of ACYABBY, the cis-elements of its promoter region were studied. Many types of regulatory elements were found in the putative promoter of ACYABBY, including elements related to stress response, hormone, reproduction, circadian rhythm control, and other regulatory mechanisms. MeJA and ABA regulate the growth and development of plants, while regulating the defense of plants against trauma, disease, infiltration, and other adverse environmental factors. 47,48 The expression levels of ACYABBYS have been reported to be distinct from that of other plants. For instance, CRC and INO are only in the reproductive development stage of Ataliaana, Bienertia rapa, and B. sinuspersici, disclosing their conservative functions in carpel morphogenesis, pistil polarization, florals meristem, and ectodermal development. 17,49 In P. granatum, PgCRC is strongly expressed in leaves, androgynous flowers, functional stamens, and skin, especially in leaves and androgynous flowers, while the flower-specific gene, PgINO, is strongly expressed only in androgynous flowers and exocarp; it is only weakly expressed in roots and exocarp. 17 In addition, previous studies have shown that FIL, YABBY2, and YABBY3 are related to the distal domain of leafderived organs, such as cotyledons, leaves, and flowers. 8,12 The FIL/YABBY3-like YABBY gene has been proven to be involved in the maintenance of meristem function. 50,51 OsYABBY4 is defined as the branch of FIL/YABBY3, which may take part in the vascular system of rice because of its dominant position in the phloem. 52 The homologous genes, AcYABBY5 and AcYABBY6, related to FIL in carambola have no specific expression regions. 53 In tomatoes, the FAS gene is similar to the YABBY2 and is crucial to the size and shape of the fruit. 54 Similar to FAS, the expression of the YABBY2-like gene in reproductive organs of carambola was noted to be higher than that in the vegetative organs. YABBY1 is similar to YABBY5, which is highly expressed in the flowers and fruits of carambola during diverse progressive stages; it may be involved in the development of carambola fruit. The main active ingredients in P. grandiflorus are terpenoids, namely the P. grandiflorus saponin, which is an important active ingredient in P. grandiflorus and has high medicinal value. The secondary metabolism 55 of plants plays a very important role in plant growth and development, adaptation to external environmental changes, biological interactions, and stress responses. Secondary metabolites are not only the result of plants adapting and evolving to suit their environment, but are also essential components of most medicinal plants. Glands and glandular hair 55 are important organs for plant secondary metabolite production; they can synthesize, secrete, and store secondary metabolites. Transcription factors 53 can regulate the secondary metabolism of plants. According to previous research, YABBY transcription factors have dual functions and can act as activators and repressors of secondary metabolites. Terpenoids are multifunctional compounds in plant secondary metabolites. Similarly, phenolic compounds are also a kind of representative secondary metabolites with unique physiological functions. Anthocyanins are the most common flavonoids amongst the phenolic compounds. In A. thaliana, FIL can positively regulate anthocyanins, 56 especially the accumulation of anthocyanins induced by JAZ protein in tissues, depending on the activity of the YABBY gene. In spearmint, MsYABBY5 can reverse regulate terpenes in plants. 55 AaYABBY5 in Artemisia annua has a positive regulatory effect on artemisinin (sesquiterpene lactones). 57 The regulation of terpenoids by YABBY genes has not been studied, and the production of secondary metabolites by plants is also related to abiotic and biotic stress factors. The abiotic stress treatment of P. grandiflorus was carried out to analyze the expression pattern of the YABBY genes in response to salt and drought stress. This provided a molecular basis for further study on the regulation of terpenoid secondary metabolites by YABBY transcription factors from the perspective of increasing the content of medicinally active components in P. grandiflorus by genetic engineering. Such investigation will also help achieve the goal of reducing research and development and the production costs of drugs related to terpenoidbased active components. The MsYABBY5 55 and AaYABBY5 of the YABBY5 subfamily have negative and positive regulatory effects on terpenoids, respectively, therefore we speculate that the PgYABBY of the YABBY5 subfamily plays a regulatory role in Platycodon species.
Salt stress inhibited the expression of PgYABBY6, while it showed a positive response in roots and leaves, indicating that PgYABBY6 was probably involved in the regulation mechanism of P. grandiflorus against salt stress. Drought only inhibited the expression of PgYABBY1 and PgYABBY2; with the extension of drought treatment time, PgYABBY3 in roots increased significantly, indicating that PgYABBY3 could respond positively to severe drought stress in roots. It could be used as a candidate gene in studying the drought resistance mechanism of P. grandiflorus roots. After 48 h of treatment, PgYABBY5 was up-regulated in the stems and leaves, proving that proper drought stress could increase the expression of this gene. PgYABBY6 responded positively to both salt and drought stress, which is of great significance to further explore the abiotic stress resistance mechanism of P. grandiflorus.

Conclusion
In this study, six YABBY genes were identified in P. grandiflorus, which is in line with the characteristics of small gene families. The characteristics of YABBY genes are conservative, and the number of introns is roughly (6 or 7). All YABBY proteins contain conservative motifs, and the number of amino acids is between 180 and 300 aa. There are many elements related to hormones and stress upstream of CsYABBY and PgYABBY at 2000 bp, indicating the expression of YABBY transcription factors in different plants. Through the genomewide identification and analysis of the YABBY transcription factor in P. grandiflorus, the influence and function of the YABBY transcription factor on the growth and development of P. grandiflorus plant organs have been predicted. It is speculated that these genes are involved in the regulation of terpene secondary metabolites, which is helpful to further studying the function of YABBY genes. Based on salt and drought stress, the expression analysis of the YABBY gene in P. grandiflorus can lay a foundation for further exploring the mechanism of the YABBY gene response to abiotic stress.